EMA生物药品分析方法验证指南

7 Westferry Circus ● Canary Wharf ● London E14 4HB ● United Kingdom Telephone +44 (0)20 7418 8400 Facsimile +44 (0)20 7418 8613 E-mail info@ema.europa.eu Website www.ema.europa.eu An agency of the European Union © European Medicines Agency, 2011. Reproduction is authorised provided the source is acknowledged. 21 July 2011 EMEA/CHMP/EWP/192217/2009 Committee for Medicinal Products for Human Use (CHMP) Guideline on bioanalytical method validation Draft agreed by the Efficacy Working Party September 2009 Adoption by CHMP for release for consultation 19 November 2009 End of consultation (deadline for comments) 31 May 2010 Agreed by Pharmacokinetics Working Party (PKWP) June 2011 Adoption by CHMP 21 July 2011 Date for coming into effect 1 February 2012 Keywords CHMP, EMEA, Guideline, validation, bioanalytical method, analyses Guideline on bioanalytical method validation Table of contents 1. Introduction (background)...................................................................... 3 2. Scope....................................................................................................... 3 3. Legal basis .............................................................................................. 3 4. Method validation.................................................................................... 4 4.1. Full validation of an analytical method................................................................... 4 4.1.1. Selectivity ................................................................................................ 5 4.1.2. Carry-over ............................................................................................... 5 4.1.3. Lower limit of quantification ........................................................................ 6 4.1.4. Calibration curve ....................................................................................... 6 4.1.5. Accuracy .................................................................................................. 7 4.1.6. Precision .................................................................................................. 7 4.1.7. Dilution integrity ....................................................................................... 8 4.1.8. Matrix effect ............................................................................................. 8 4.1.9. Stability ................................................................................................... 9 4.2. Partial validation .............................................................................................. 10 4.3. Cross validation ............................................................................................... 10 5. Analysis of study samples ..................................................................... 10 5.1. Analytical run .................................................................................................. 11 5.2. Acceptance criteria of an analytical run ............................................................... 11 5.3. Calibration range ............................................................................................. 12 5.4. Reanalysis of study samples .............................................................................. 12 5.5. Integration...................................................................................................... 13 6. Incurred samples reanalysis.................................................................. 13 7. Ligand binding assays ........................................................................... 14 7.1.1. Full validation ......................................................................................... 14 7.2. Partial validation and cross-validation ................................................................. 17 7.3. Analysis of study samples.................................................................................. 17 7.3.1. Analytical run ......................................................................................... 17 7.3.2. .Acceptance criteria for study sample analysis ............................................. 17 7.3.3. Incurred samples reanalysis...................................................................... 18 8. Reports.................................................................................................. 18 8.1. Validation report .............................................................................................. 18 8.2. Analytical report .............................................................................................. 19 Definitions................................................................................................. 20 Page 2/22 Executive summary This guideline defines key elements necessary for the validation of bioanalytical methods. The guideline focuses on the validation of the bioanalytical methods generating quantitative concentration data used for pharmacokinetic and toxicokinetic parameter determinations. Guidance and criteria are given on the application of these validated methods in the routine analysis of study samples from animal and human studies. 1. Introduction (background) Measurement of drug concentrations in biological matrices (such as serum, plasma, blood, urine, and saliva) is an important aspect of medicinal product development. Such data may be required to support applications for new actives substances and generics as well as variations to authorised drug products. The results of animal toxicokinetic studies and of clinical trials, including bioequivalence studies are used to make critical decisions supporting the safety and efficacy of a medicinal drug substance or product. It is therefore paramount that the applied bioanalytical methods used are well characterised, fully validated and documented to a satisfactory standard in order to yield reliable results. Acceptance criteria wider than those defined in this guideline may be used in special situations. This should be prospectively defined based on the intended use of the method. 2. Scope This guideline provides recommendations for the validation of bioanalytical methods applied to measure drug concentrations in biological matrices obtained in animal toxicokinetic studies and all phases of clinical trials. As ligand binding assays differ substantially from chromatographic analytical methods, separate validation recommendations for ligand binding assays are provided. In addition, specific aspects for the analysis of study samples will be addressed. Furthermore, this guideline will describe when partial validation or cross validation should be carried out in addition to the full validation of an analytical method. Methods used for determining quantitative concentrations of biomarkers used in assessing pharmacodynamic endpoints are out of the scope of this guideline. 3. Legal basis This guideline has to be read in conjunction with the introduction and general principles (4) and Part I and II of the Annex I to Directive 2001/83 as amended. It applies to Marketing Authorisation Applications for human medicinal products submitted in accordance with the Directive 2001/83/EC as amended, and Regulation (EC) No. 726/2004, in which the analysis of drug concentrations in a biological matrix is part of the application. The validation of bioanalytical methods and the analysis of study samples for clinical trials in humans should be performed following the principles of Good Clinical Practice (GCP). Further guidance that will help clinical laboratories develop and maintain quality systems which will comply with relevant European Union Directives, national regulations and associated guidance documents can be found in the “Reflection Paper for Laboratories That Perform The Analysis Or Evaluation Of Clinical Trial Samples.” (EMA/INS/GCP/532137/2010). Page 3/22 Non-clinical (pharmaco-toxicological) studies submitted in a marketing authorisation application shall be carried out in conformity with the provisions related to Good Laboratory Practice, Directive 2004/10/EC on the harmonisation of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances and Directive 2004/9/EC on the inspection and verification of good laboratory practice (GLP). Normally, the validation of bioanalytical methods used in non-clinical pharmacotoxicological studies that are carried out in conformity with the provisions related to Good Laboratory Practice should be performed following the Principles of Good Laboratory Practice. Aspects of method validation not performed according to GLP should be clearly identified and their potential impact on the validation status of the method indicated. Methods used in pre-clinical studies not required to be performed to GLP should be fit for purpose but not necessarily developed in a GLP facility. 4. Method validation 4.1. Full validation of an analytical method A full method validation should be performed for any analytical method whether new or based upon literature. The main objective of method validation is to demonstrate the reliability of a particular method for the determination of an analyte concentration in a specific biological matrix, such as blood, serum, plasma, urine, or saliva. Moreover, if an anticoagulant is used, validation should be performed using the same anticoagulant as for the study samples. Generally a full validation should be performed for each species and matrix concerned. In some cases, it may be problematic for validation purposes to obtain an identical matrix compared to the matrix of the study samples. A suitable alternative matrix may be used, e.g. synthetically prepared cerebrospinal fluid, if justified. The main characteristics of a bioanalytical method that are essential to ensure the acceptability of the performance and the reliability of analytical results are: selectivity, lower limit of quantification, the response function and calibration range (calibration curve performance), accuracy, precision, matrix effects, stability of the analyte(s) in the biological matrix and stability of the analyte(s) and of the internal standard in the stock and working solutions and in extracts under the entire period of storage and processing conditions. Usually one analyte or drug has to be determined, but on occasions it may be appropriate to measure more than one analyte. This may involve two different drugs, but can also involve a parent drug with its metabolites, or the enantiomers or isomers of a drug. In these cases the principles of validation and analysis apply to all analytes of interest. Reference standards During method validation and analysis of study samples, a blank biological matrix will be spiked with the analyte(s) of interest using solutions of reference standard(s) to prepare calibration standards, quality control samples and stability samples. In addition, suitable internal standard(s) (IS) can be added during sample processing in chromatographic methods. It is important that the quality of the reference standard and IS is ensured, as the quality (purity) may affect the outcome of the analysis, and therefore the outcome of the study data. Therefore the reference standards used during the validation and study sample analysis should be obtained from an authentic and traceable source. Suitable reference standards, include certified standards such as Page 4/22 compendial standards (EPCRS, USP, WHO), commercially available standards, or sufficiently characterised standards prepared in-house or by an external non-commercial organisation. A certificate of analysis is required to ensure purity and provide information on storage conditions, expiration date and batch number of the reference standard. The use of certified standards is not needed for IS, as long as the suitability for use is demonstrated, e.g. lack of analytical interference is shown for the substance itself or any impurities thereof. A certificate of analysis is not required. When mass-spectrometry (MS) detection is used in the bioanalytical method, a stable isotope-labelled IS is recommended to be used whenever possible. However, it is essential that the labelled standard is of the highest isotope purity and that no isotope exchange reaction occurs. The presence of any unlabelled analyte should be checked and if relative amounts of unlabelled analyte are detected the potential influence has to be evaluated during method validation. 4.1.1. Selectivity The analytical method should be able to differentiate the analyte(s) of interest and IS from endogenous components in the matrix or other components in the sample. Selectivity should be proved using at least 6 individual sources of the appropriate blank matrix, which are individually analysed and evaluated for interference. Use of fewer sources is acceptable in case of rare matrices. Normally, absence of interfering components is accepted where the response is less than 20% of the lower limit of quantification for the analyte and 5% for the internal standard. It may also be necessary to investigate the extent of any interference caused by metabolites of the drug(s), interference from degradation products formed during sample preparation, and interference from possible co-administered medications. Co-medications normally used in the subject population studied which may potentially interfere should be taken into account at the stage of method validation, or on a study specific and compound specific base. The possibility of back-conversion of a metabolite into parent analyte during the successive steps of the analysis (including extraction procedures or in the MS source) should also be evaluated, when relevant (i.e. potentially unstable metabolites e.g. acidic metabolites to ester, unstable N-oxides or glucuronide metabolites, lactone-ring structures). The extent of back-conversion should be established and the impact on the study results discussed. It is acknowledged that this evaluation will not be possible early during drug development of a new chemical entity when the metabolism is not yet evaluated. However, it is expected that this issue is taken into account and a partial validation is performed if relevant as further knowledge regarding metabolism of the active substance is gained during drug development. It is recognized that in some cases it is very difficult to obtain the metabolites of interest. Alternatively, back-conversion of a metabolite can be checked by applying incurred sample reanalysis. However, in this case potential back conversion during sample processing cannot be ruled out. 4.1.2. Carry-over Carry-over should be addressed and minimised during method development. During validation carry- over should be assessed by injecting blank samples after a high concentration sample or calibration standard at the upper limit of quantification. Carry over in the blank sample following the high concentration standard should not be greater than 20% of the lower limit of quantification (LLOQ; see below) and 5% for the internal standard. If it appears that carry-over is unavoidable, study samples should not be randomised. Specific measures should be considered, tested during the validation and Page 5/22 applied during the analysis of the study samples, so that it does not affect accuracy and precision. This could include the injection of blank samples after samples with an expected high concentration, before the analysis of the next study sample. 4.1.3. Lower limit of quantification The lower limit of quantification (LLOQ) is the lowest concentration of analyte in a sample which can be quantified reliably, with an acceptable accuracy and precision. The LLOQ is considered being the lowest calibration standard (see Accuracy and Precision). In addition, the analyte signal of the LLOQ sample should be at least 5 times the signal of a blank sample. The LLOQ should be adapted to expected concentrations and to the aim of the study. As an example, for bioequivalence studies the LLOQ should be not higher than 5% of the Cmax, while such a low LLOQ may be not necessary for exploratory pharmacokinetic studies. 4.1.4. Calibration curve The response of the instrument with regard to the concentration of analyte should be known, and should be evaluated over a specified concentration range. The calibration standards should be prepared in the same matrix as the matrix of the intended study samples by spiking the blank matrix with known concentrations of the analyte. There should be one calibration curve for each analyte studied in the method validation and for each analytical run. Ideally, before carrying out the validation of the analytical method it should be known what concentration range is expected. This range should be covered by the calibration curve range, defined by the LLOQ being the lowest calibration standard and the upper limit of quantification (ULOQ), being the highest calibration standard. The range should be established to allow adequate description of the pharmacokinetics of the analyte of interest. A minimum of six calibration concentration levels should be used, in addition to the blank sample (processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS). Each calibration standard can be analysed in replicate. A relationship which can simply and adequately describe the response of the instrument with regard to the concentration of analyte should be applied. The blank and zero samples should not be taken into consideration to calculate the calibration curve parameters. The calibration curve parameters should be reported (slope and intercept in case of linear fit). In addition, the back calculated concentrations of the calibration standards should be presented together with the calculated mean accuracy values (see definition of Accuracy below). All the available (or acceptable) curves obtained during validation, with a minimum of 3 should be reported. The back calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of the calibration standards, with a minimum of six calibration standard levels, must fulfil this criterion. In case replicates are used, the criteria (within ±15% or ±20% for LLOQ) should also be fulfilled for at least 50% of the calibration standards tested per concentration level. In case a calibration standard does not comply with these criteria, this calibration standard sample should be rejected, and the calibration curve without this calibration standard should be re-evaluated, including regression analysis. In case all replicates of the LLOQ or the ULOQ calibration standard are rejected then the batch should be rejected from the validation, the possible source of the failure be determined and the method revised (if necessary). If the next validation batch also fails, then the method should be revised before restarting validation. Page 6/22 Although the calibration curve should preferably be prepared using freshly spiked samples, it is allowed to use previously prepared and stored calibration samples, if supported by appropriate stability data. 4.1.5. Accuracy The accuracy of an analytical method describes the closen